PTRF-IL33-ZBP1 signaling mediating macrophage necroptosis contributes to HDM-induced airway inflammation

Polymerase 1 and transcript release factor (PTRF, encoding by Cavin-1) regulates interleukin 33 (IL-33) release, which is implicated in asthma development. Z-DNA binding protein 1 (ZBP1)-sensing Z-RNAs induces necroptosis which causes inflammatory diseases. House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma. Whether PTRF via IL-33 and ZBP1 mediates HDM-induced macrophage necroptosis and airway inflammation remains unclear. Here, we found that deficiency of PTRF could reduce lung IL-33, ZBP1, phosphor-receptor-interacting protein kinase 3 (p-RIPK3), and phosphor-mixed lineage kinase domain-like (p-MLKL) (necroptosis executioner), and airway inflammation in an HDM-induced asthma mouse model. In HDM-treated macrophages, ZBP1, p-RIPK3, and p-MLKL levels were markedly increased, and these changes were reversed by deletion of Cavin-1. Deletion of Il33 also reduced expression of ZBP1, p-RIPK3, and p-MLKL in HDM-challenged lungs. Moreover, IL-33 synergizing with HDM boosted expression of ZBP1, p-RIPK3, and p-MLKL in macrophages. In bronchial epithelial cells rather than macrophages and vascular endothelial cells, PTRF positively regulates IL-33 expression. Therefore, we conclude that PTRF mediates HDM-induced macrophage ZBP1/necroptosis and airway inflammation, and this effect could be boosted by bronchial epithelial cell-derived IL-33. Our findings suggest that PTRF-IL33-ZBP1 signaling pathway might be a promising target for dampening airway inflammation.


INTRODUCTION
Asthma is a widespread airway disorder leading to wheezing, shortness of breath, chest tightness, and cough. Asthma is characterized by chronic airway inflammation, triggering processes such as airway hyperresponsiveness (AHR), mucus production, and remodeling of the airway wall. Asthma is a common chronic disease among both adults and children in the United States, affecting 25 million people and resulting in nearly one-half million hospitalizations annually [1]. Only some patients respond well to the medications and strategies currently used in the clinic. Asthma results from a complex interaction between structural and immune cells after exposure to specific environmental triggers [2]. Therefore, further understanding the mechanism underlying asthma may allow physicians to decide the best treatment for asthmatic patients.
Polymerase 1 and transcript release factor (PTRF), coded by Cavin-1, is a cytoplasmic protein containing a putative leucine zipper, a nuclear localization signal, and a PEST (amino acid sequence enriched in proline (P), glutamic acid (E), serine (S), and threonine (T)) domain [3]. Ubiquitously expressed in multiple tissues, including the lung, PTRF was initially identified as regulating transcription by interacting with RNA polymerase 1 and dissociating the paused transcription complex involving transcription termination factor 1 (TTF-1). PTRF has been suggested to be an essential structural component of caveolae. Caveolae are vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment [4]. Moreover, accumulating evidence has shown that caveolae are present in various cells in the lung and interact with other proteins, receptors, and ion channels, potentially affecting normal and disease processes such as contractility, inflammation, and fibrosis [5]. PTRF participates in some key pathways during the progression of many lung diseases. For example, PTRF plays a role in receptor tyrosine kinases (RTK)-mediated pro-survival signaling in lung adenocarcinomas [6], also PTRF is a protein biomarker for chronic obstructive pulmonary disease (COPD) [7]. In an ovalbumin (OVA)induced asthma mouse model, PTRF is reported to have an inhibitory effect on IL-33 release [8].House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma [9]. However, whether PTRF is involved in the development of HDM-induced allergic airway inflammation, and the detailed mechanisms remain unknown.
As we know, IL-33 contributes to the activation of type 2 immunity cells, such as group 2 innate lymphoid cells (ILC2s), T helper 2 cells (Th2), macrophages, and eosinophils in the development of asthma. IL-33 is also an important proinflammatory cytokine released from necrotic cells. Our previous study found that PTRF phosphorylation status regulated IL-33 release and eventually affected asthma exacerbation [8]. Necroptosis directly induces the release of full-length biologically active IL-33 in an inflammatory disease model and in vitro [15]. Whether IL-33 affects ZBP1-medited necroptosis is unknown.
Therefore, the objectives of this study are to determine: (i) whether deficiency of PTRF would attenuate HDM-induced airway inflammation; (ii) Whether deficiency of PTRF affects Zbp1 expression and necroptosis executioners (p-RIPK3 and p-MLKL), IL-33 in HDM-challenged lungs; (iii) Whether deficiency of PTRF affects ZBP1, p-RIPK3, and p-MLKL levels in HDM-challenged macrophages; and (iv) whether IL-33 synergizes with HDM to boost ZBP1, p-RIPK3, and p-MLKL signaling in HDM-challenged macrophages. Ultimately, we will identify whether PTRF-IL33-ZBP1 signaling would mediate necroptosis in macrophages, which contributes to HDM-induced airway inflammation.

Deficiency of PTRF ameliorates HDM-induced airway inflammation
The genotyping protocols to identify Cavin-1 +/+ , Cavin-1 +/-, and Cavin-1 -/mice and confirmation of PTRF expression at both mRNA and protein levels in these mice were shown in Supplementary Fig.  1A-C. Cavin-1 -/mice suffered from very low birth rate and severe growth problems, so we only obtained 3 homozygotes during 3 years of breeding. These 3 Cavin-1 -/mice were used for HDMchallenged lung RNAseq analysis. Alternatively, Cavin-1 +/mice were used in our routine experiments. To investigate the potential role of PTRF in regulating airway inflammation induced by HDM, we established a mouse asthma model by intranasal administrations of HDM (Fig. 1A). In Cavin-1 +/+ mice, HDM treatment significantly increased total cells and protein in bronchoalveolar lavage fluid (BALF) compared with PBS-treated mice (Fig. 1B, C). We also found plasma immunoglobulin E (IgE) was increased in the asthma model (Fig. 1D). BALF inflammatory parameters and plasma IgE antibody titers were reduced in HDM-treated Cavin-1 +/− mice compared with HDM-treated Cavin-1 +/+ littermates. Airway resistance, an indicator of AHR, was also significantly decreased in HDM-treated Cavin-1 +/− mice compared with HDM-treated Cavin-1 +/+ littermates (Fig. 1E). Further histopathological examination of lung sections with hematoxylin and eosin (HE) and periodic acid-schiff (PAS) staining revealed that peribronchial inflammatory cell infiltration and mucus expression were reduced in HDM-treated Cavin-1 +/− mice compared to HDM-treated Cavin-1 +/+ mice (Fig. 1F, G). Lung IL-33 at both mRNA and protein levels were decreased in HDM-treated Cavin-1 +/− mice compared to HDM-treated Cavin-1 +/+ mice (Fig. 1H, I). The findings suggest that PTRF is causative factor for the development of HDM-induced airway inflammation and positively regulates IL-33 expression.
To study time-dependent effect of HDM challenge, we then treated Raw264.7 with HDM at 50 μg/ml for 0, 12, 24, and 36 h. We found that HDM increased the mRNA levels of Cavin-1, Zbp1, and necroptosis-related genes Ripk3 and Mlkl at 24 h after HDM challenge. The same pattern was also in Tnfα, Il6, and Il1b expression (Fig. 4D). Consistent with mRNA analysis, HDM treatment also increased the expression of PTRF and ZBP1, phosphorylation of RIPK3 and MLKL at 24 h (Fig. 4E), but did affect cleavage of GSDMD and pro-IL-1β (Fig. 4F). These findings confirm that HDM is able to trigger necroptosis in macrophages by upregulating necrotizing signaling pathway molecules.
We repeated the above experiments in bronchial epithelial cells (BEAS-2B) and vascular endothelial cells (HUVEC) using the same experimental protocols. We found that HDM challenge could increase IL-33 expression at concentration of 50 and 100 μg/ml in bronchial epithelial cells. Knockdown of Cavin-1 could reduce IL-33 expression in HDM-treated bronchial epithelial cells, suggesting that PTRF positively regulates IL-33 expression in HDM-challenged I IL-33 protein levels were detected by ELISA in lung homogenates of Cavin-1 +/+ and Cavin-1 +/mice. Each point represents an individual mouse. B, C, D, H, I, data were means ± SD with n = 4-7 mice in per group. G data were means ± SD with n = 4 mice in per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated by two-tailed unpaired student's t test, corrected by one-way ANOVA with Turkey post-hoc test. Fig. 2A, B). HDM and knockdown of Cavin-1 did not alter IL-33 expression in HUVEC ( Supplementary Fig. 2C, D). HDM and knockdown of Cavin-1 did not affect expression of PTRF and ZBP1, and p-RIPK3 and p-MLKL in both bronchial epithelial and vascular endothelial cells either (Supplementary Fig. 2A-D).
Knockdown of Zbp1 in Raw264.7 macrophages attenuates HDM-triggered necroptosis signaling We further explored whether ZBP1 would regulate necroptosis in vitro. We built up 3 Zbp1 shRNA to silence Zbp1 in Raw264.7 macrophages. More than 50% ZBP1 was deleted at both mRNA and protein levels using the NO.3 construct of Zbp1 shRNA (Fig. 6A, B). We treated the Raw264.7 macrophages with the NO.3 construct of Zbp1 shRNA and its scrambled counterpart, followed by challenging the cells with HDM. By RT-PCR analysis, we found that necroptosis-related Ripk1, Ripk3, Tnfα, Il6, and Il1b at mRNA levels were significantly upregulated in HDM-challenged scrambled shRNA transfected cells. In contrast, these genes were markedly reduced in HDM-challenged Zbp1 shRNA transfected cells compared to HDM-challenged scrambled shRNA transfected cells (Fig. 6C). We observed that necroptosis molecules ZBP1 and p-MLKL were upregulated in scrambled shRNA transfected cells following HDM stimulation; however, this change was reversed in Zbp1 shRNA transfected cells (Fig. 6D). We also found that cell death induced by HDM stimulation was reduced in Zbp1 shRNA transfected cells compared to scrambled shRNA transfected cells with PI staining (Fig. 6E). LDH was also increased in HDMchallenged scrambled shRNA transfected cells, and this change was also attenuated in HDM-challenged Zbp1 shRNA transfected cells (Fig. 5F). Taken together, these results imply that ZBP1 regulates necroptosis in HDM-challenged macrophages.
HDM and IL-33 synergistically increases p-RIPK3 and p-MLKL in Raw264.7 macrophages and BMDMs We treated Raw264.7 macrophages and BMDMs from WT mouse with either PBS or IL-33, followed by PBS or HDM challenge. We found that IL-33 + HDM treated group could significantly increase ZBP1, p-RIPK3, and p-MLKL compared to IL-33 or HDM-treated group (Fig. 9A, C). However, HDM + IL-33 treatment did not affect cleavage of GSDMD, pro-IL-1β, and caspase 3 (Fig. 9B, D). These findings suggest that HDM and IL-33 synergistically increases promotes necroptosis in macrophages by upregulating p-RIPK3 and p-MLKL. In summary, as shown in Fig. 9E, PTRF positively regulates HDM-induced IL-33 expression in bronchial epithelial cells. Concurrently, PTRF increases ZBP1 expression and necroptosis signaling in HDM-challenged macrophages. Furthermore, bronchial epithelial cell-derived IL-33 works synergistically with HDM to enhance ZBP1 expression and necroptosis in HDMchallenged macrophages. The above processes contribute to airway inflammation.

DISCUSSION
In this study, we first demonstrated that deficiency of Cavin-1 could attenuate HDM-induced airway inflammation. We then found that Zbp1 expression and necroptosis executioners (p-RIPK3 and p-MLKL), and IL-33 were significantly decreased in Cavin-1deficient HDM-challenged lungs. In macrophages, HDM challenge . D Zbp1, Ripk1, Ripk3, and Mlkl mRNAs were assessed by RT-qPCR (normalized to GAPDH). E Tnfα, Il6, and Il1β mRNAs were assessed by RT-qPCR (normalized to GAPDH). F Lactate dehydrogenase (LDH) activity measured at an optical density of 490 nm (OD490) in BALF samples (n = 3-5 mice). G Western blot analysis of PTRF, ZBP1 and necroptosis components in the lungs collected from PBS or HDM treated Cavin-1 +/+ and Cavin-1 +/mice. Necroptosis activation was indicated by the phosphorylation of receptor interacting protein kinase 3 (p-RIPK3) and mixed lineage kinase domain-like pseudokinase (p-MLKL). H Western blot analysis of pyroptosis and apoptosis activation markers after HDM exposure in the lungs collected from PBS or HDM treated Cavin-1 +/+ and Cavin-1 +/mice. Pyroptosis activation was assessed by immunoblotting of cleaved GSDMD (35 kDa) and mature IL-1β (17 kDa). Apoptosis activation was determined by immunoblotting of executioner caspase3 (17 kDa). IL-33 in the lung tissues was also detected with immunoblotting. In (G, H), tubulin was used as a loading control for immunoblot analysis and molecular weight marker sizes were indicated on the right (kDa). Data were from three independent experiments (n = 3-6 mice in each group) and represented as means ± SD. *P < 0.05, **P < 0.01 as calculated by two-tailed unpaired student's t test, corrected by one-way ANOVA with Turkey post-hoc test.  Fig. 5 Knockdown of Cavin-1 in Raw264.7 macrophages attenuates HDM-triggered ZBP1/necroptosis signaling. A The Raw264.7 cells treated with scrambled shRNA or Cavin-1 shRNA were stimulated with HDM (50 μg/ml) for 24 h. B Raw264.7 were transfected with either scrambled or Cavin-1 shRNAs and Western blot was performed for the protein expression of PTRF in cell lysates. C Cavin-1, Zbp1, Ripk1, Ripk3, Mlkl, Tnfα, Il6 and Il1b mRNAs were assessed by RT-qPCR (normalized to GAPDH). D Western blot analysis of PTRF, ZBP1 and necroptosis components in cell lysates. Necroptosis activation was indicated by p-RIPK3 and p-MLKL. E Real-time analysis of cell death in Raw 264.7 using the PI staining after treated with HDM for 24 h. The original magnification is ×10. Quantification of mean fluorescence intensity of PI staining cells. F LDH activity measured at OD490 in cell supernatant. Data were means ± SD and n = 3 biological replicates in per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated by two-tailed unpaired student's t test, corrected by one-way ANOVA with Turkey posthoc test.  Fig. 6 Knockdown of Zbp-1 in Raw264.7 macrophages attenuates HDM-triggered necroptosis signaling. A The Raw264.7 cells treated with scrambled shRNA or Zbp-1 shRNA were stimulated with HDM (50 μg/ml) for 24 h. B Raw264.7 were transfected with either scrambled or Zbp-1 shRNAs and Western blot was performed for the protein expression of ZBP1 in cell lysates. C Cavin-1, Zbp1, Ripk1, Ripk3, Mlkl, Tnfα, Il6 and Il1b mRNAs were assessed by RT-qPCR (normalized to GAPDH). D Western blot analysis of PTRF, ZBP1 and necroptosis components in cell lysates. Necroptosis activation was indicated by p-RIPK3 and p-MLKL. E Real-time analysis of cell death in Raw 264.7 using the PI staining after treated with HDM for 24 h. The original magnification is ×10. Quantification of mean fluorescence intensity of PI staining cells. F LDH activity measured at OD490 in cell supernatant. Data were means ± SD and n = 3 biological replicates in per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated by two-tailed unpaired student's t test, corrected by one-way ANOVA with Turkey post-hoc test.
increased ZBP1, p-RIPK3, and p-MLKL levels, and these changes could be reversed by genetic knockdown or deletion of Cavin-1. More importantly, we found that ZBP1, p-RIPK3, and p-MLKL were reduced in Il33-deficient HDM-challenged lungs. PTRF positively regulates IL-33 in bronchial epithelial cells. In presence of IL-33, ZBP1, p-RIPK3, and p-MLKL were significantly increased in HDMchallenged macrophages.
PTRF, coded by Cavin-1, is a critical constituent of the caveolae structure on the plasma membrane [4]. Caveolae are flask-shaped invaginations of the plasma membrane present in most structural cells. The lungs express numerous caveolae and high levels of PTRF, which play important roles in pulmonary diseases such as lung cancer and pulmonary hypertension [6,17]. Expression of PTRF in the airway smooth muscle is increased in an OVA-induced asthma model [18]. Partial loss of PTRF led to a greater AHR and potent type 2 immune responses during challenge phase of OVAinduced asthma model, without influencing the sensitization phase. Knockdown of PTRF in 16HBE led to a significantly increased level of IL-33 in cell culture supernatants in response to LPS or HDM [8]. In this study, we found that deletion of Cavin-1  (G, H), tubulin was used as a loading control for immunoblot analysis and molecular weight marker sizes were indicated on the right (kDa). Data are means ± SD and n = 3-5 mice in each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated by two-tailed unpaired student's t test, corrected by one-way ANOVA with Turkey post-hoc test. and BMDMs from WT mouse (C, D) were treated with either PBS or IL-33 (rmIL-33, 50 μg/ml), and followed by either PBS or HDM (50 μg/ml) for 24 h. The cells were harvested for Western blotting analysis. A, C Western blot analysis of IL-33, ZBP1, and necroptosis components p-RIPK3, p-MLKL in cell lysates of Raw264.7 and BMDMs. B, D Western blotting analysis of pyroptosis markers cleaved GSDMD, mature IL-1β and apoptosis executioner cleaved caspase3 after HDM/rmIL-33 exposure in cell lysates. GAPDH was used as an internal control for immunoblot analysis and molecular weight marker sizes were indicated on the right (kDa). n = 3 biological replicates in each group. E PTRF positively regulates HDM-induced IL-33 expression in bronchial epithelial cells. Concurrently, PTRF increases ZBP1 expression and necroptosis signaling in HDM-challenged macrophages. Furthermore, bronchial epithelial cell-derived IL-33 works synergistically with HDM to enhance ZBP1 expression and necroptosis in HDM-challenged macrophages. Thus, the above effects contribute to HDM-induced macrophage necroptosis and airway inflammation.
reduces HDM-induced airway inflammation and lung IL-33 at both mRNA and protein levels ( Figs. 1 and 3H). Knockdown of Cavin-1 also reduces IL-33 in HDM-induced BEAS-2B bronchial epithelial cells detected by Western blotting (Supplementary Fig. 2). These findings suggest that the modulating effect of PTRF on inflammation and IL-33 expression may depend on different cell lines or inducers of inflammation. It is reported that necroptosis directly induces the release of nuclear IL-33 in its full-length form in an Aspergillus extractinduced asthma model. Aspergillus extract could trigger necroptosis and IL-33 release in L929 or HaCaT cells [15]. Here, we found that HDM can increase IL-33 expression in bronchial epithelial cells (Supplementary Fig. 2B) rather than macrophages (Fig. 7E) and vascular endothelial cells (Supplementary Fig. 2D). HDM could not induce necroptosis in the bronchial epithelial cells ( Supplementary  Fig. 2A). Hence, we speculate that HDM might work with the other proinflammatory cytokines (TNF-α, etc) to induce necroptosis and IL-33 release in the airway epithelial cells of HDM-challenged asthma mouse model. We have demonstrated that HDM-treated macrophages could produce TNF-α, and this effect could be attenuated by knockdown or deletion of Cavin-1 (Figs. 5C and 7A). This notion was supported by airway epithelial cell necroptosis occurs in HDM-induced allergic inflammation mouse model [19].
Macrophages form the first line of defense against microbes and airborne particles through multiple functions, including phagocytosis, production of cytokines and chemokines, and antigen presentation. Emerging studies suggest ZBP1 is a pathogen sensor (for DNA and RNA) that regulates of cell death and inflammatory responses [20]. ZBP1 is abundantly expressed in macrophages and contributes to necroptosis [21]. ZBP1, which complexes with RIPK3 to trigger RIPK3-driven pathways, including trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death [22]. Interestingly, our HDM-challenged lung RNAseq analysis showed that effect of deletion of Cavin-1 on immune response and doublestranded RNA-binding genes, especially Zbp1, has the same pattern as deletion of ll33 (Figs. 3A-C and 8A-C). Western blotting analysis also showed that deletion of Cavin-1 manifested as deletion of Il33 could reduce ZBP1, p-RIPK3, and p-MLKL signaling pathway (Figs. 3G and 8G). Thus, we conclude that both PTRF and IL-33 positively regulate ZBP1-necroptosis during HDM-induced airway inflammation. More importantly, we confirmed that knockdown or deletion of Cavin-1 could reduce HDM-induced necroptosis in HDM-challenged macrophages (Figs. 5C and 7A).
In our study, we also found that airway inflammation and necroptosis markers were decreased in Il33 -/asthma mice, while co-exposure to HDM + rmIL-33 synergistically increased the ZBP1/ necroptosis in Raw264.7 cells and BMDMs. We assumed that PTRF upregulated the expression of IL-33 in bronchial epithelial cells, which may contribute to HDM-promoted inflammation through upregulating ZBP1/necroptosis signaling in macrophages. These findings may help us explain the role of PTRF in allergic inflammation in new perspectives.
A recent study demonstrates that amino-terminal p40 fragment GSDMD, whose generation was independent of inflammatory caspase-1 and caspase-11, dominates cytosolic secretion of IL-33 by forming pores in the cell membrane in A549 and MLE-12 cells (lung epithelial type II cells) [1]. In our study, we did not find that HDM could induce cleavage of GSDMD and pro-IL-1β in bronchial epithelial cells, which is inconsistent with the study reported by Ge et al. [23]. Whether HDM induces p40 GSDMD fragmentation in bronchial epithelial cells warrants further investigation. This study has several limitations. First, we conducted experiments using Cavin-1 +/mice since Cavin-1 -/mice exhibit a very low birth rate and growth problems. Second, the location of PTRF expression was unknown in our study. How did HDM regulate PTRF expression? How did PTRF regulate Zbp1 expression? These questions will be investigated in our future research. The deletion of Cavin-1 in macrophages might help us understand the role PTRF in mediating airway inflammation.
Taken together, PTRF positively regulates HDM-induced IL-33 expression in bronchial epithelial cells. Concurrently, PTRF increases ZBP1 expression and necroptosis signaling in HDMchallenged macrophages. Furthermore, bronchial epithelial cellderived IL-33 works synergistically with HDM to enhance ZBP1 expression and necroptosis in HDM-challenged macrophages. Therefore, we conclude that PTRF-IL33-ZBP1 signaling mediating macrophage necroptosis contributes to HDM-induced airway inflammation. Our findings highlight the critical role of PTRF via regulating ZBP1/necroptosis in macrophages to drive HDMinduced airway inflammation.

MATERIALS AND METHODS Animals and Asthma model
The Cavin-1 +/mice (B6.129S6-Ptrftm1Pfp/J) were kindly provided by Professor K. Liao from the Shanghai Institute of Biochemistry and Cell Biology [24]. Heterozygous mice (Cavin-1 +/-) were crossed to breed Cavin-1 +/+ and heterozygous mice. Since Cavin-1 -/mouse had a very low birth rate and growth problems and Cavin-1 +/mouse had a very low protein level of PTRF in the lung, we used Cavin-1 +/mice to perform the experiments [8]. Il33 -/mice were provided by F. Zheng from the Huazhong University of Science and Technology [25]. Mice were housed under specific-pathogen-free conditions for 12 h dark/light cycles. Mice had access to food and water ad libitum. All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of the Institute Pasteur of Shanghai, Chinese Academy of Sciences (Animal Ethics Review Number: A2018052). Female WT, Cavin-1 +/-, and Il33 -/-(6-8 wk old) mice were used for experiments. Females were randomly allocated to experimental groups and no blinding method was used for treatment. There was no animal exclusion criteria. The HDM-induced mouse asthma model was established, as described previously [26]. Mice were challenged with intranasal administrations of 35 μl [0.7 mg/ml phosphate-buffered saline (PBS)] of whole HDM protein extract (Greer Laboratory, Boston, Mass) for five consecutive days per week (days 1-5) in two weeks. Control animals received only PBS. The anesthesia was induced with an intraperitoneal (i.p.) injection of pentobarbital sodium (50 mg/kg) before the mice were sacrificed. Mice were euthanatized for analysis 24 h after the last HDM treatment. All experiments were repeated at least three times with similar sample sizes.

Isolation and culture of bone marrow-derived macrophages
Primary BMDMs were isolated and cultured as described previously [27]. Briefly, mice were sacrificed using standard CO 2 asphyxiation guidelines followed by cervical dislocation. Using an aseptic technique, bone marrow was harvested from the femur and tibia bones. The marrow cavities were flushed with RPMI 1640 medium and bone marrow was collected. After centrifuging the blood samples at 300 g for 5 min and eliminating erythrocytes, the remaining cells were resuspended in a complete macrophage culture medium (CMCM, RPMI 1640 containing 10% FBS, 20% L929 cell-conditioned medium, 100 IU/ml penicillin and 100 μg/ml streptomycin). Cells were seeded at 37°C with 5% CO 2 for 7 days and CMCM was replaced on days 3 and 5. BMDMs were collected for the experiment on day 7 of culture.

Cell culture and transfection
All cell lines were cultured in a humidified incubator at 37°C with 21% O 2 and 5% CO 2 . Raw 264.7, BEAS-2B, L929, and HEK293T cells were purchased from ATCC (American Type Culture Collection Manassas, USA) and grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 μM glutamine, 100 μg/ml streptomycin sulfate, and 100 IU/ml penicillin. HUVEC were purchased from ScienCell and cultured in an endothelial cell growth medium (ScienCell, USA) containing 100× endothelial cell growth supplement (ScienCell, USA) and 5% FBS. All cell lines were recently authenticated by STR profiling, and the mycoplasma contamination test of all cells was negative. HDM protein extract (Greer Laboratory, Boston, Mass) and recombinant Mouse IL-33 (rmIL-33, Biolegend, 580506) were used for stimulating Raw264.7 or BMDMs.

Airway resistance index
AHR was measured using the Lung Function System (AniRes2005 V3.5, Animal Pulmonary Function Analysis System, Beijing Bestlab High-Tech Co., Ltd, China) 24 h after the last treatment. Briefly, the mice were anesthetized and connected to a pressure transducer via a tracheal cannula. Increasing concentrations of methacholine (0.025, 0.05, 0.1, and 0.2 mg/kg body weight) were injected into the external jugular vein at 5-min intervals using a fine needle. Resistance of the lung (RL), resistance to expiration (Re), and dynamic respiratory compliance (Cdyn) were recorded to evaluate the airway reactivity.

Bronchoalveolar lavage fluid collection
Lavage of the lungs was performed by flushing with 1 ml PBS 3 times at the end of experiments. After centrifuging, the supernatant was used to measure LDH with LDH Cytotoxicity Assay Kit and total protein concentration by Pierce BCA assay (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The cell pellet from BALF was then resuspended in PBS and analyzed for total cell count with TC20 automated cell counter (Bio-Rad Inc, Hercules, California). The remaining BALF cells were stained for Flow cytometric analysis.

Isolation of mouse lung cells
After anesthetization, mice were performed tracheal intubation, and then blood was taken by exsanguinating mice from the Vena Cava. Blood plasma was performed for IgE measurement. After BALF was collected, 1 ml of dispase II (2 U/ml) was injected through the trachea [28]. Subsequently, the lungs were incubated and digested in 2 ml of 2 μg/ml collagenase/dispase II containing 0.001% DNAse 1 for 30 min at 37°C on a shaker. Cells were collected by centrifugation at 335 × g for 10 min at 4°C in a 15 ml conical tube. ACK lysis buffer was used for lysing erythrocytes, and the cells were rinsed twice with cold PBS/0.5% BSA. After being resuspended in 1 ml cold PBS/0.5% BSA, single cells were passed through a 70 μm cell strainer and collected for flow cytometry. Total cells were diluted and counted by the TC20 automated cell counter.

Flow cytometry
The methods of flow cytometry adhere to the guidelines [29]. BALF cells and lung cells were prepared as described previously. For cell surface staining, cell suspensions were incubated with the antibody cocktails for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated with Leukocyte Activation Cocktail for 4 h (BD Pharmingen, 550583), which contained 50 ng/ml phorbol12-myristate 13-acetate (PMA), 1 μg/ml ionomycin and 1 μg/ml GolgiPlugTM protein transport inhibitor. Then, Cytofix/ Cytoperm Kit (BD Pharmingen, 554714) was used to intracellular cytokines staining. For nuclear protein staining, cells were treated with Transcription Factor Buffer Set (BD Pharmingen, 562574) according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay and LDH release assay
Whole lungs were homogenized in 1 ml PBS containing 0.05% Triton X-100, Pierce Protease and Phosphatase Inhibitor cocktail (Thermo Scientific™). Suspensions were filtered with a 40 μm cell strainer and clarified by centrifugation. Supernatant was used for detecting cytokines by ELISA [30]. The levels of IL-4, IL-5, IL-13, IL-33, and IgE were measured with sandwich ELISA kit according to the manufacturer's instructions. For IL-33 in lung homogenate, also total protein determination was performed from the same sample, and the results were then presented in relation to total protein concentration for each sample. LDH release was detected in BALF and cells supernatant using the LDH Cytotoxicity Assay Kit (88953; Thermo Scientific) according to the manufacturer's instructions. Readings were carried out at a 490 nm wavelength, using a microplate reader (Thermo Scientific) and expressed as % LDH release.

Propidium iodide staining
Cells were cultured in 12-well plates, stimulated with PBS or HDM for 24 h, washed twice with cold PBS, and then levitated in 1 mL binding